Dihydrofolate reductase (DHFR) and Thymidylate Synthase (TS) are two key enzymes for DNA synthesis and represent some of the most important targets for cancer chemotherapy. DHFR catalyzes the reduction of folate and 7, 8 dihydrofolate to 5, 6, 7, 8 tetrahydrofolate, the latter as the one-carbon donor is essential for the formation of thymidylate (dTMP) which is the precursor for DNA synthesis (see Banerjee et al., “Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase, Biochim. Biophys. Acta. (2002) 1587: 164-173). TS catalyzes the reductive methylation of dUMP by CH2H4folate to produce dTMP and H2folate (see Carreras et al., “The catalytic mechanism and structure of thymidylate synthase,” Annu. Rev. Biochem. (1995) 64: 721-762). DHFR inhibitors, such as methotrexate (MTX), and TS inhibitors, such as 5-fluorouracil (5-FU) and Tomudex or Ralitrexed (TDX, ZD1694), are widely used chemotherapeutic drugs for the treatment of osteosarcoma and colon cancer (see Widemann et al., “Understanding and managing methotrexate nephrotoxicity,” Oncologist. (2006) 11: 694-703; see also Calvert, “An overview of folate metabolism: features relevant to the action and toxicities of antifolate anticancer agents,” Semin. Oncol. (1999) 26: 3-10).
As such, TS and DHFR are the major targets of cancer chemotherapy in the clinic today. TDX, the third-generation TS inhibitor, is an active agent in the treatment of human colon and breast cancer (see Drake et al., “Resistance to tomudex (ZD1694): multifactorial in human breast and colon carcinoma cell lines,” Biochem. Pharmacol. (1996) 51: 1349-1355). The inhibitor of DHFR, such as MTX, is widely used in the treatment of human leukemia, osteosarcoma and choriocarcinoma. Increased DHFR protein levels are reported to be associated with drug resistance (see Banerjee et al., “Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase,” Biochem. Biophys. Acta. (2002) 1587: 164-173), and low tumor expression levels of TS have also been linked with improved outcome for colon cancer patients treated with 5-FU chemotherapy (see Soong et al., “Prognostic significance of thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase protein expression in colorectal cancer patients treated with or without 5-fluorouracil-based chemotherapy,” Ann. Oncol. (2008) 19: 915-919). However, MTX has the highest activity at the time when DNA synthesis, DHFR activity, DHFR content, and DHFR mRNA content increased and the lowest activity at the time when they decreased (see Yamauchi et al., “Ohdo S. Cell-cycle-dependent pharmacology of methotrexate in HL-60,” J. Pharmacol. Sci. (2005) 99: 335-341).
Thus, there exists a need for better prognostic and diagnostic measures, treatment and control of neoplasm through application of small molecules to target cells to affect various cellular components, such as TS and DHFR involved in cellular proliferation of neoplasia.